Review
MicroRNAs in liver cancer: a model for investigating
pathogenesis and novel therapeutic approaches
E Callegari
1, L Gramantieri
2, M Domenicali
3, L D’Abundo
1, S Sabbioni*
,4and M Negrini*
,1MicroRNAs (miRNAs) constitute a large class of short RNAs (e.g., 20–24 nucleotides in length), whose main function is to
posttranscriptionally regulate the expression of protein-coding genes. Their importance in tumorigenesis has been
demonstrated over the past decade, and correspondingly, they have emerged as potential therapeutic molecules and targets.
Liver cancer is one of the most common neoplastic diseases worldwide, and it currently has a poor prognosis owing to largely
ineffective therapeutic options. Liver cancer is also an excellent model for testing miRNA-based therapy approaches as it can be
easily targeted with the systemic delivery of oligonucleotides. In recent years, the role of miRNAs in hepatocellular carcinoma
(HCC) has been established with molecular studies and the development of animal models. These studies have also provided the
basis for evaluating the therapeutic potential of miRNAs, or anti-miRNAs. In general, the safety of miRNAs has been proven and
antitumor activity has been observed. Moreover, because of the absence or presence of mild side effects, the prophylactic use of
miRNA-based approaches may be foreseen.
Cell Death and Differentiation (2015) 22, 46–57; doi:10.1038/cdd.2014.136; published online 5 September 2014
Facts
Hepatocellular carcinoma (HCC) is a primary malignant
disease of the liver with poor prognosis.
With the exception of the multi-kinase inhibitor, sorafenib,
there are no effective systemic treatments available for
HCC.
Several miRNAs are differentially expressed in HCC and
may be pathogenically relevant.
Genetically modified animal models have been developed
to demonstrate the direct role of miRNAs in the initiation
and/or progression of cancer.
The in vivo therapeutic efficacy of certain miRNA mimics or
anti-miRNA oligonucleotides has been evaluated.
Open Questions
Do the animal models that are available faithfully represent
the etiology and natural history of human HCC?
Are methods for the in vivo systemic delivery of miRNAs/
anti-miRNAs sufficiently developed, and can they be
optimized?
Are miRNA/anti-miRNA molecules effective against liver
cancer?
What are the clinical settings in which miRNA/anti-miRNA
molecules may be successfully applied?
MicroRNAs (miRNAs) constitute a large class of short RNAs
(e.g., 20–24 nucleotides in length), which have key roles in cell
development and differentiation by mediating the
post-transcriptional regulation of protein-coding genes.
1Over the
past decade, the critical role of miRNAs in tumorigenesis has
been widely investigated and their important regulatory action
in several biological processes that are frequently altered in
cancer has been described. At present, miRNAs have a
well-recognized role in human carcinogenesis, including
hepato-carcinogenesis, and accumulating experimental evidence
indicates that they may act as oncogenes or tumor suppressor
genes.
2,3It is also well known that the expression of several
miRNAs is deregulated in human HCC compared with normal
tissue.
4,5In this context, miRNAs have also emerged as novel
molecules or targets for tumor therapy, and liver cancer
represents an excellent model for their testing.
HCC is the most common primary liver malignant disease,
and the third cause of cancer-related deaths, worldwide.
6It has a poor prognosis and the therapeutic options currently
available are largely ineffective. Molecular studies have
revealed several pathogenic mechanisms at the basis of liver
cancer. For example, HCC pathogenesis involves multiple
1
Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Universita` di Ferrara, 44121 Ferrara, Italy;
2Centro di Ricerca Biomedica Applicata e Dipartimento di
Medicina Interna, Policlinico S. Orsola-Malpighi e Universita` di Bologna, 40138 Bologna, Italy;
3Unita` di Semeiotica Medica, Policlinico S. Orsola-Malpighi, Dipartimento
di Scienze Mediche e Chirurgiche, Universita` di Bologna, 40138 Bologna, Italy and
4Dipartimento di Scienze della Vita e Biotecnologie, Universita` di Ferrara, 44121
Ferrara, Italy
*Corresponding authors: S Sabbioni, Dipartimento di Scienze della Vita e Biotecnologie, Universita` di Ferrara, 44121 Ferrara, Italy. Tel: +39 0532 455319;
Fax: +39 0532 455875; E-mail: [email protected]
or M Negrini, Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Universita` di Ferrara, 44121 Ferrara, Italy. Tel: +39 0532 455399; Fax: +39 0532 455875;
E-mail: [email protected]
Received 17.3.14; revised 02.7.14; accepted 24.7.14; Edited by RA Knight; published online 05.9.14
Abbreviations: HCC, hepatocellular carcinoma; HCV, hepatitis C virus; miRNA, microRNA; DEN, N-nitrosodiethylamine; HGF, hepatocyte growth factor; CDK,
cyclin-dependent kinase; IAP, inhibitor of apoptosis protein; ROCK2, rho-cyclin-dependent kinase; AAV, adeno-associated virus; CRAds, conditionally replicating adenovirus
genetic and epigenetic alterations that lead to the deregulation
of several signaling pathways, including the p53, PI3K/Akt/
mTOR, Ras/Raf/MEK/ERK, IGFR, and Wnt/b-catenin
path-ways (for reviews, see Gramantieri et al.
7and Aravalli et al.
8).
Recent studies using next-generation sequencing technology
have further refined our knowledge of the signaling pathways
involved in liver carcinogenesis.
9–11Regulation of these
cellular signaling pathways by miRNAs indicates that this
class of short RNAs may present a new set of potentially
therapeutic tools for HCC.
12,13miRNAs and HCC
Several studies have revealed that the expression of certain
miRNAs is deregulated in human HCC compared with
matched nonneoplastic tissue (for a comprehensive review,
see Negrini et al.
14). Moreover, molecular and cellular studies
have established that aberrantly expressed miRNAs can
affect crucial cancer-associated pathways.
2,3Cell proliferation and survival. The discovery of
modu-lated gene targets has helped recognize the contribution
of miRNAs to cancer-associated pathways. Unrestricted
cellular proliferation and prolonged survival have critical
roles in the process of hepatocarcinogenesis. In the cell cycle
context, abnormal expression of miRNAs has been shown to
alter the functionality of several cell cycle regulators,
including RB1, cyclin-dependent kinases (CDKs), cyclins,
and CDK inhibitors. RB1 is a target of miR-335.
15miR-335 is
downregulated during mesenchymal stem cell
differentia-tion
16and upregulated by the Wnt signaling pathway,
16which is often activated in HCC. RB1 is also a target of
miR-221, a miRNA that is frequently overexpressed in HCC.
17,18These observations suggest that expression of RB1, a key
G1–S cell cycle transition checkpoint protein, may be
aberrantly regulated by altered miRNA expression. Among
the other proteins that control cell cycle progression
(Figure 1), cyclins are reported to be upregulated in HCC,
whereas negative cell cycle regulators are often
down-regulated compared with surrounding parenchyma.
19Over-expression of cyclinD1/CDK4 has been detected in
B60% of
HCC cases.
20Among CDK inhibitors, p16/INK4A is
function-ally inactivated owing to deletions in the short arm of
chromosome 9 in about 20% of HCCs
21or by promoter
methylation in 30–70% of HCC cases.
22All of these genes
are also reported to be affected by altered expression of
miRNAs (Figure 1). For example, miR-124 and miR-203,
which inhibit the growth of HCC cells via downregulation of
CDK6,
23are methylated and silenced in HCC cell lines and
primary tumors.
23Direct targeting of cyclin D1, CDK6, and
E2F3 by miR-195 can also block the G1–S transition,
whereas inhibition of miR-195 promotes cell-cycle
progres-sion.
24MiR-195 can also suppress the ability of HCC cells to
form colonies in vitro, and the development of tumors in nude
mice. In most HCC tissues and cell lines, miR-195
expres-sion is reduced,
24and this may contribute to the upregulation
of cyclinD1/CDK that is observed in HCC. MiR-26 is another
miRNA whose expression is reduced in HCC,
25thereby
preventing its induction of cell cycle arrest in liver cancer cells
via the direct targeting of cyclins D2 and E2.
26Members of
the KIP family of CDK inhibitors, CDKN1A/p21, CDKN1B/
p27, and CDKN1C/p57, also act as tumor suppressors in
HCC
by
negatively
affecting
cell-cycle
progression.
CDKN1B/p27 and CDKN1C/p57 proteins are downregulated
in HCC compared with surrounding cirrhosis.
27One
mechan-ism that leads to a reduction in CDKN1C/p57 expression is
the loss of maternal allele methylation at the
KvDMR1-imprinted locus at 11p15.5. This has been found in 20–50%
of HCCs.
28Another mechanism involved in the
downregula-tion of p27 and p57 is the overexpression of miR-221/222,
which occurs in about 70–80% of HCCs.
18Owing to their
inhibition of RB1 expression as well, levels of miR-221/222
appear to be major factors in cell-cycle control. CDKN1A/p21
is downregulated by oncogenic members of the miR-17-92
family.
29–33Among the three clusters of the miR-17-92 family
of the human genome, those on chromosomes 13 and 7 are
upregulated in HCC.
34Disruption of apoptosis in HCC has been extensively
reviewed.
35,36In this context, miRNAs contribute to the
altered expression of members of the BCL2 family
(Figure 2). For example, upregulation of antiapoptotic
members may result from reduced expression of miRNAs,
with miR-122 controlling BCL-W and BCL-XL
37while the latter
is also controlled by let-7 members.
38MCL1 is a target of
miR-101,
39miR-193b,
40and miR-29,
41,42and all of these miRNAs
are downregulated in HCC. Conversely, proapoptotic
mem-bers may be inhibited by overexpressed miRNAs. For
example, the BH3-only proteins, BMF and BID, are targeted
by miR-221 and miR-25, respectively.
29,30,43These
mechan-isms have the potential to allow miR-221 to protect cells from
‘anoikis’, a form of apoptosis induced by the detachment of
anchorage-dependent cells from the surrounding extracellular
matrix. MiR-25 may also impair the TGF-b tumor suppressor
pathway. Both pathways represent critical steps in the
process of metastasis.
44,45miR-483 is a miRNA located
within intron 2 of the IGF2 gene, and it is highly expressed in
fetal liver, yet is barely detectable in adult liver.
46Moreover, it
is overexpressed in 30–40% of HCCs either independently or
in combination with IGF2. MiR-483-3p has also been shown to
promote cell survival by repressing translation of the
p53-inducible BH3-only protein, PUMA/BBC3.
47Apoptosis is also controlled by molecules not belonging to
the BCL2 family. For example, the inhibitor of apoptosis
protein (IAP), survivin, is overexpressed in HCC and is
controlled by miR-203,
48a miRNA silenced by aberrant DNA
methylation in HCC tissue.
23Another miRNA, miR-21,
appears to promote cell survival under stressful conditions.
Indeed, miR-21 is upregulated in HCC, as well as in many
other human neoplasms, and is associated with poor
prognosis
49,50and resistance to chemotherapy.
51–53These
effects appears to be linked to the survival advantage
imparted by miR-21 via the direct targeting of proapoptotic
genes, such as the tumor suppressor lipid-phosphatase,
PTEN, which controls the phosphatidyl inositol 3-phosphate
kinase (PI3K) signaling pathway
54–56and programmed cell
death 4 (Pdcd4) protein.
53,57–62The latter also has a role in
TGF-b-induced apoptosis.
At the nexus of cell cycle and apoptosis signaling is a
molecular pathway that is controlled by the tumor-suppressor
protein, p53. Genetic alterations in the TP53 gene have been
Figure 1
Aberrant miRNA expression in liver cancer promotes cell-cycle progression. A simplified pathway of cell cycle regulation is presented that shows the effects of various aberrantly expressed miRNAs in liver cancer. Upregulated miRNAs are indicated with a red up arrow, and downregulated miRNAs are indicated with a green down arrow. Upregulated miRNAs exert a stronger inhibitory effect on their targets, whereas downregulated miRNAs mediate weaker effects on their targets. Taken together, multiple aberrant miRNAs act on this pathway to support cell cycle progressionFigure 2
Aberrant miRNA expression in liver cancer affects the functionality of apoptosis pathways. A simplified apoptosis pathway is presented that shows the effects of various miRNAs that are aberrantly expressed in liver cancer. The aberrant expression of multiple miRNAs supports cell survivalextensively described in HCC,
21,63,64and execution of the p53
pathway in HCC is affected by miRNA expression at various
levels (Figure 2). For example, the loss of miR-122 expression
in HCC cells leads to repression of p53 via upregulation of
cyclin G1.
65,66MiR-145 also induces p53 activity by an
unknown mechanism,
67and is commonly downregulated in
HCC.
65In many cases, miRNAs that are downstream
effectors of p53 are downregulated owing to p53 loss of
function. Although not fully proven in HCC, this situation is
exemplified by miR-34a that is downregulated in some HCCs,
most likely owing to the presence of a nonfunctional p53.
However, in other HCCs, miR-34a is upregulated.
54,68Lack of
miR-34a induction can promote several biological effects
through increased expression of CDK4, cyclin E2, BCL2, and
the tyrosine kinase receptor, MET.
69–74Other examples of
p53-inducible miRNAs include miR-145, whose lack of
expression leads to the activation of MYC,
75or the cluster,
miR-192/215.
76,77Similar to 34, either 145 or
miR-192/215 can promote cell-cycle arrest and/or apoptosis,
colony
suppression,
and
can
increase
CDKN1A/p21
levels.
67,76,77Invasion and metastasis. Advanced tumor features, such
as the ability of cancer cells to promote uncontrolled
angiogenesis and to invade tissues and blood vessels, are
also affected by miRNA deregulation. One of the signaling
pathways that confers invasive potential to HCC cells is
mediated by the HGF/MET axis. Hepatocyte growth factor
(HGF) binds to its transmembrane tyrosine kinase receptor,
MET, and promotes hepatocyte proliferation, migration,
survival, and angiogenesis. HGF also mediates invasive
growth
during
embryonic
development
and
tumori-genesis.
78,79Overexpression of MET is found in 40–70% of
HCCs
80–83and is regulated by miR-199-3p (indicated earlier
as miR-199a*), miR-34a, miR-23b, and miR-1.
84–88All these
miRNAs are downregulated in HCC, thereby contributing to
the upregulation of c-MET. Among the downstream effectors
of RTKs, the overexpression of RAS, but rarely activating
point mutations, has been demonstrated in HCC.
89,90All
members of the RAS family have been shown to be
modulated by various members of the let-7 family, and the
latter are downregulated in HCC,
65as well as in several other
human cancers,
91–97thus suggesting that let-7 potentially
contributes to the upregulation of RAS.
As previously mentioned, the PI3K/AKT/PTEN pathway
promotes cell survival as well as invasion and metastasis.
Correspondingly, the targeting of PTEN by 21 and
miR-221 can favor an invasive phenotype.
98,99Indeed, the
silencing of PTEN and PDCD4 by miR-21 has resulted in a
decrease in apoptosis and an increase in cell invasion.
MiR-21, which is upregulated in HCC and in most human
malignancies, is linked to invasion and metastasis via its
targeting of multiple tumor/metastasis suppressor genes,
including tropomyosin 1 (TPM1), maspin, tissue inhibitor of
metalloproteinase 3 (TIMP3) gene, and RHOB.
54,100–103In
addition, miR-21-mediated inhibition of RECK, a
membrane-anchored glycoprotein that negatively regulates matrix
metal-loproteinase-9, leads to increased cell invasion.
98In the
context of the PI3K/AKT pathway, activation of the mTOR
pathway has been found to be common to several human
cancers, including HCC.
104Moreover, when it is
over-expressed, it is associated with poor prognosis, invasion,
and metastasis.
105Despite this, a very low rate of genetic
alterations that affect the mTOR pathway in HCC has been
reported.
104Recently, mTOR was identified as a direct target
of 3p, and to be inversely correlated with
miR-199a-3p in HCC.
84The miR-199/214 cluster is of particular interest
as it is downregulated in the majority of HCCs that have been
studied,
4,54,65,84,106,107as well as in other human
malignan-cies,
108,109in cancer-derived cell lines,
110and in experimental
neoplastic and pre-neoplastic conditions.
111Other deregulated miRNAs that have a role in the invasive
and metastatic properties of HCC cells include 122,
miR-139, and miR-151. For example, loss of miR-122 enhances
cell migration and invasion, and its restoration in metastatic
liver cancer cells has been shown to significantly reduce
migration, invasion, and anchorage-independent growth
in vitro, as well as tumorigenesis, angiogenesis, and
intrahepatic metastasis in vivo.
66,112,113Invasive and
meta-static properties of HCC are also linked to the loss of control of
ADAM17 by miR-122. Indeed, specific silencing of ADAM17
resulted in a dramatic reduction in migration and invasion
in vitro, and reduced tumorigenesis, angiogenesis, and local
invasion in the livers of nude mice.
114These observations are
similar to those associated with the restoration of miR-122.
Downregulation of miR-122, which has been detected in about
70% of HCCs,
5,54,65is also associated with the development
of intrahepatic metastases
114and a shorter time to
recur-rence.
66Reduced expression of miR-139 has also been
detected in HCC,
106,115and has specifically been linked to the
acquisition of metastatic properties.
115In HCC cells,
over-expression of miR-139 has significantly reduced cell migration
and invasion in vitro, and the incidence and severity of lung
metastases in vivo. These effects were linked to interactions
between miR-139 and the 3
0untranslated region of rho-kinase
2 (ROCK2). In human metastatic HCC, expression of miR-139
is reduced and levels of miR-139 inversely correlate with
levels of ROCK2 protein in human HCC samples.
115MiR-151
is frequently amplified and overexpressed in HCC in
combination with its host gene, focal adhesion kinase
(FAK).
116It is possible that the host gene, FAK, cooperates
with miR-151 to enhance cell motility and spreading effects,
and this would suggest that miR-151 has a critical role in tumor
invasion and metastasis. MiR-151-5p targeting of RhoGDIA, a
putative metastasis suppressor, has also been found to
increase HCC cell migration and invasion, as well as the
activation of Rac1, Cdc42, and Rho GTPases.
116The b-catenin pathway has a central role in the
develop-ment of liver cancer and is an essential component of both
intercellular junctions and canonical Wnt signaling. Thus,
b-catenin is important for the regulation of cell proliferation,
differentiation, and stemness
19,117–121(Figure 3). In HCC, the
Wnt/b-catenin pathway has been found to be abnormally
activated through gain-of-function mutations at the
N-termi-nus of b-catenin (present in 12–26% of HCCs),
21by deletions,
mutations, or epigenetic alterations of the E-cadherin gene,
and by loss-of-function mutations in the AXIN1 or AXIN2
genes (present in 8–13% of HCCs).
21,122The capacity for
nuclear b-catenin to promote the epithelial to mesenchymal
transition has been shown to induce invasive and metastatic
properties of tumor cells.
123Correspondingly, the silencing of
b-catenin reverses the epithelial–mesenchymal transition and
represses metastatic potential.
124In a recent review of
miRNA regulation of the Wnt/b-catenin pathway,
125it is
apparent that miR-21, miR-200, miR-315, and miR-135
directly regulate the Wnt/b-catenin core. MiR-135 and
miR-315 activate b-catenin by inhibiting the negative regulators,
APC and axin, respectively.
126,127MiR-200a has been shown
to regulate b-catenin levels either directly
128or indirectly by
modulating ZEB1/2, and this downregulation of miR-200a in
HCC has been reported in various studies,
4,65,106,129,130thereby supporting its role in mediating an increase in nuclear
b-catenin levels and the activation of the pathway in cancer
cells. MiR-34a has also been shown to be a negative regulator
of the Wnt pathway based on its targeting of WNT1.
131,132In
HCC where miR-34 is downregulated, repression of WNT1 is
released. Moreover, in addition to invasion and metastasis,
the Wnt/b-catenin pathway is also important for maintaining
stemness.
118–120In this context, it has been proposed that
upregulation of miR-181b supports this function at two levels,
by repressing the Wnt/b-catenin inhibitor, NLK, and by
blocking the hepatic differentiation transcription factors,
CDX2 and GATA6,
133suggesting that liver cancer cells may
arise from stem cells that have lost control over their
self-renewal potential.
As described above, miRNAs appear to have an essential
role in the modulation of complex cross-talk that exists
between
pathways
affecting
HCC
development
and
progression. Their understanding may facilitate the
develop-ment of novel, targeted therapeutic strategies against HCC.
Animal Models of Liver Cancer
The importance of deregulated miRNAs in malignant cell
transformation and tumor development has been observed in
several studies. In particular, the development of animal
models that are genetically modified at miRNA loci have
proven the involvement of miRNAs in the initiation and
progression of cancers, and provided in vivo models to test
the efficacy of miRNA-based therapeutic approaches. Most of
the models that have been developed are related to
hematopoietic diseases, but HCC models have also been
developed (Table 1).
miRNA-specific models of HCC. For HCC, various cancer
genes, including tumor suppressor genes, oncogenes, and
hepatitis B virus or hepatitis C virus (HCV) viral genes, have
been employed for the development of mouse models.
134–144More recently, miRNA-based mouse models predisposed to
HCC have been reported. A transgenic mouse model
characterized by overexpression of miR-221 in the liver
was developed. In these mice, male animals exhibit a strong
predisposition for HCC, which includes the emergence of
spontaneous nodular liver lesions with age and a strong
acceleration of tumor development after treatment with the
carcinogen,
N-nitrosodiethylamine
(DEN).
Notably,
the
Figure 3
Aberrant miRNA expression in liver cancer supports the activation of various signaling pathways. Simplified diagrams of RAS/MAPK, PI3K/AKT/mTOR, and WNT/b-catenin signaling pathways are shown, along with the effects of various miRNAs that are aberrantly expressed in liver cancer. As a result, cell proliferation, survival, and maintenance of stemness are enhancedTG221 mouse was the first transgenic animal model to
develop a solid tumor following overexpression of a
miRNA.
145Among the miRNAs that are downregulated in
HCC, two studies have reported the development of miR-122
knockout mice. These mice are characterized by hepatic
inflammation, fibrosis, and the development of spontaneous
liver tumors with age. These studies also demonstrated the
tumor suppressor function of miR-122 in the liver, as well as
its
importance
in
liver
metabolism
and
hepatocyte
differentiation.
146,147In addition to these miRNA-specific models, the importance
of deregulated miRNA machinery components has also
recently been suggested. For DDX20, a DEAD box protein
component of miRNA-containing ribonucleoprotein
com-plexes, it suppresses NF-kB activity by regulating miR-140
function.
148Correspondingly, miR-140 knockout mice
149are
prone to HCC after treatment with DEN compared with control
animals, and they have a phenotype that is similar to that for
DDX20 deficiency. Taken together, these results suggest that
miR-140 may act as a tumor suppressor by regulating the
NF-kB pathway.
149These reports confirm the importance of
miRNA deregulation in liver cancer.
Traditional HCC preclinical models. In addition to
geneti-cally modified mouse models, traditional preclinical models of
liver cancer have been established with chemical treatment.
There are two main methods of HCC induction using
chemicals: (i) administration of carcinogens alone, such as
DEN, over a prolonged period of time or by intraperitoneum
injection,
150or (ii) administration of carcinogens (CCl
4and
DEN) followed by a promotion phase (partial hepatectomy) or
administration of alcohol or phenobarbital.
151,152DEN is
frequently used as a carcinogenic agent;
152however, the
target organ in which it induces malignant tumors is species
specific. Thus, mice treated with DEN develop liver tumors as
well as gastrointestinal,
153skin, lung,
154and haematopoietic
malignancies.
155In addition, the time needed to develop HCC
after a single DEN injection depends on the administered
dose, and the gender, age, and strain of the treated mice.
156Other models of chemically induced liver disease involve
the administration of CCl
4in drinking water, by inhalation, or
by subcutaneous or intraperitoneal injections.
151,157CCl
4treatment has been shown to induce an advanced stage of
liver cirrhosis and related complications, such as ascites
decompensation, in both rats and mice.
157,158However,
development of HCC has not been reported. In a two-stage
chemical model of HCC, CCl
4administration is accompanied
by alcohol supplementation in drinking water.
159However,
only after 104 weeks does this combination induce the
development of HCC and metastases.
miRNA-Based Therapies
A comparative analysis of global gene expression patterns for
murine and human HCC has shown that many models can
reproduce key biological and molecular events observed in
the human condition.
160Accordingly, mouse models have
been widely used for the testing of potential therapeutic
targets and for preclinical studies (see Li et al.
161for a recent
review). The efficacy of miRNA-mediated HCC prevention
and therapy has also been evaluated in recent years using
various strategies involving miRNA inhibition or miRNA
replacement.
miRNA inhibition. Krutzfeldt and colleagues were the first
to demonstrate that miRNAs could be effectively silenced in
mice using specific molecules. In this study, the liver-specific
expression of miR-122 was inhibited with an intravenous
administration of chemically modified single-stranded RNA,
known as ‘antagomirs’, that were complementary to the
target miRNA.
162The safety of this silencing approach was
established in nonhuman primates, where no evidence of
liver toxicity was detected.
163On the basis of these
promising results, many studies of miRNA inhibition were
subsequently performed. The tumor-promoting activity of
miR-221 in HCC was also demonstrated in vivo. In one
notable study, systemic delivery of a cholesterol-tagged
anti-miR-221 to orthotopic HCC tumors was found to reduce
tumor cell proliferation and promote survival.
164In a second
study, anti-miR-221 oligonucleotides were found to exhibit
significant antitumor activity in the TG221 transgenic mouse
model, where a significant reduction in the number and size
of tumors in the treated mice was confirmed with
histopatho-logical analyses. Significant downregulation of miR-221
levels has also been detected in liver tissues of
anti-miR-221-treated mice, thereby confirming the ability of these
molecules to inhibit endogenous miR-221
145(Table 2). The
importance of manipulating miRNA levels as a therapeutic
tool for treating liver cancer is also supported in the work by
Lim et al.,
143where anti-miR inhibition of miR-494 (a miRNA
Table 1 miRNA-based animal modelsmiRNA Mouse model name Mouse model Tumor
miR-155 E(mu)-mmu-miR155 Transgenic B-cell malignancies miR-155 mir-155LSLtTA Knockin B-cell malignancies miR-29a/b Emu-miR-29 Transgenic B-cell malignancies
miR15a/16-1 DLEU2/miR15a/16-1 Knockout Chronic lymphocytic leukemia miR-21 mir-21LSL-Tetoff Knockout B-cell malignancies
miR-125b Emu/miR-125b Transgenic B-cell malignancies miR-146a miR-146a KO Knockout Myeloproliferative disease miR-17B92 Emu-miR-17B92 Transgenic B-cell malignancies miR-122 Mir122-LKO Knockout Hepatocellular carcinoma miR-122 Mir122a–/– Knockout Hepatocellular carcinoma miR-140 MiRNA-140 / Knockout Hepatocellular carcinoma
overexpressed in human HCC) resulted in a significant
reduction in tumor size in a primary MYC-driven liver tumor
mouse model.
HCV promotes HCC by inducing chronic liver disease and
cirrhosis. MiR-122 has been found to support HCV
replica-tion.
165–167Correspondingly,
treatment
of
chronically
infected nonhuman primates with an LNA molecule
comple-mentary to the miR-122 sequence led to a long-lasting and
well-tolerated suppression of HCV viremia. These results
support the hypothesis that miR-122 represents a potential
target for antiviral intervention and possible liver disease
sequels.
168,169Currently, an anti-miR-122 oligonucleotide,
‘miravirsen SPC3649’ (Santaris Pharma, A/S, Hørsholm,
Denmark), is being evaluated in a phase 2 clinical trial for the
treatment of HCV infection (ClinicalTrials.gov Identifier
NCT01200420)
170(Figure 4). However, the concomitant
inhibition of the tumor-suppressor activity of miR-122
65,146,147must also be considered.
miRNA replacement. The miRNA restoration is another
strategy that has been used to elucidate the functions of
miRNAs, to identify their roles in tumorigenesis, and to
provide preclinical tools for the treatment of cancer. One
anticancer approach involves the restoration of miRNAs that
are generally downregulated in cancer cells. In vivo xenograft
models have been used to test the tumor suppressor activity
of several miRNAs that were delivered by adeno-associated
viruses (AAV), nano-sized lipid particles, or by
cholesterol-conjugated modified oligonucleotides (Table 2). Hou et al.
171used both cholesterol-conjugated small RNAs and an AAV
delivery system to effectively restore miR-199a/b-3p
expres-sion in HCC tissues of HCC-bearing nude mice. As a result,
tumor growth was inhibited without evidence of toxicity.
Lentivirus-mediated expression of miR-199a was also able to
inhibit tumor growth in a nude mouse xenograft model,
172thereby confirming the antitumor effect of this miRNA in HCC
and its therapeutic potential.
To provide a better model of human HCC, genetically
engineered or chemically induced mouse models have been
employed. For example, using MYC-driven mouse models of
HCC, the tumor suppressor activity of miR-26a
26and
miR-122
146have been demonstrated. In a DEN-induced HCC
mouse model, the systemic administration of miR-124 led to a
reduction in tumor growth and tumor size via induction of
apoptosis and deregulation of a miRNA/inflammatory
feed-back loop circuit involving hepatocyte nuclear factor 4alpha
(HNF4a).
173Taken together, these studies indicate that
miRNA mimics represent a promising approach for the
treatment of cancer, and could be translated into clinical
practice. In fact, MRX34 (Mirna Therapeutics, Inc., Austin, TX,
USA), a liposome-formulated mimic of the tumor suppressor
miR-34a, is the first miRNA mimic that has entered a
multicenter phase I clinical trial aimed at evaluating safety in
patients with primary liver cancer or with liver metastasis from
other cancers (ClinicalTrials.gov Identifier: NCT01829971)
174(Figure 4).
Oncolytic viruses. A distinct approach that has become
interlaced with miRNA expression in HCC is the field of
oncolytic viruses. These vectors have tumor-specific activity
that results in the cytolysis of cancer cells, whereas normal
tissues exhibit minimal toxicity in vivo. Thus, the use of
oncolytic viruses represents a promising approach for the
treatment of cancer (for a recent review, see Patel et al.
175and Callegari et al.
176). The differential expression of
miRNAs in neoplastic versus normal tissues has also been
used to improve the safety of oncolytic virus-based therapies.
For example, to overcome liver toxicity associated with the
systemic delivery of oncolytic adenoviruses, regulatory
miRNA sequences have been inserted in the genome of
conditionally replicating adenoviruses (CRAds). In one case,
CRAds regulated by miR-122 were generated to provide
liver-specific suppression of viral replication without
compro-mising the replication capacity of the modified virus in cancer
Figure 4
miRNA-based approaches that are moving toward clinical trials. An overview of the many preclinical studies that have produced promising results, as well as the clinical trials that are being planned or are underwaytissues in vivo.
177–179Other uses of oncolytic viruses have
included the generation of a let-7-dependent adenovirus that
is able to replicate in tumor cells, and not in normal liver cells,
in a HCC xenograft model.
180Similarly, a
miR-199-depen-dent CRAd was generated to replicate in HCC tumor cells
and not in normal liver cells. This virus was able to control
tumor growth in a subcutaneous xenograft model in nude
mice, and in HCCs arising in immune-competent mice,
without causing significant hepatotoxicity
181(Table 2).
Conclusions
HCC is the most common type of liver cancer diagnosed, and
treatment options currently depend on tumor size and staging.
For patients with advanced, non-resectable HCC, the only
systemic therapy available involves the multi-kinase inhibitor,
sorafenib.
182,183This molecule targets the tyrosine kinase
receptors VEGFR and PDGFR, as well as the serine–
threonine kinases c-RAF and BRAF. Randomized phase 3
trials with sorafenib have shown an improvement in median
overall survival and progression-free survival for HCC
patients.
182,184However, more effective treatments that have
less toxic side effects are still needed. The use of miRNAs or
anti-miRNAs
as
antitumor
therapeutic
molecules
has
attracted a lot of attention. At present, their short-term safety
has been proven in several instances, albeit their efficacy as
anticancer agents remains to be fully confirmed. Various
studies have demonstrated that miR-26a, miR-199, miR-122,
anti-221, and 494 reduce tumor growth, and
miR-34a has entered a clinical trial. For anti-miR-122, its capacity
to inhibit HCV replication has the potential to prevent the
development of HCC. Future investigations may also focus on
the use of miRNA/anti-miRNA oligonucleotides for the
prevention of HCC owing to the limited presence, or absence,
of side effects associated with these approaches. To this
end, animal models that are predisposed to HCC represent
important instruments for testing the effectiveness of miRNA/
anti-miRNA molecules.
Animal models of HCC have been established and are
widely
used
for
preclinical
and
translational
studies.
Moreover, the study of several HCC murine models has
provided a comprehensive characterization of the molecular
mechanisms of liver carcinogenesis and has also resulted in
the development of new therapeutic strategies.
185Compared
with human HCC, genetically modified mouse models
gen-erally develop HCC without the development of liver cirrhosis.
However, this condition is present in 480% of human HCC
cases. The administration of CCl
4has been shown to induce
advanced stages of liver cirrhosis, although this is not
accompanied by the development of HCC.
157Therefore,
CCl
4treatment of genetically modified mice may lead to the
development of models that more closely represent the
human condition where HCC develops in cirrhotic livers.
Future studies will be needed to investigate these possibilities.
Acknowledgements. This work was supported by funding from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Italian Ministry of University and Research, the University of Ferrara, and by the Fondazione Cassa di Risparmio di Bologna (Carisbo).
1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
2. Negrini M, Ferracin M, Sabbioni S, Croce CM. MicroRNAs in human cancer: from research to therapy. J Cell Sci 2007; 120(Pt 11): 1833–1840.
3. Gailhouste L, Ochiya T. Cancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma. Histol Histopathol 2013; 28: 437–451. 4. Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T et al.
Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006; 25: 2537–2545.
5. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 2008; 47: 1955–1963. 6. Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence and
mortality rates and trends. Cancer Epidemiol Biomarkers Prev 2010; 19: 1893–1907. 7. Gramantieri L, Fornari F, Callegari E, Sabbioni S, Lanza G, Croce CM et al. MicroRNA
involvement in hepatocellular carcinoma. J Cell Mol Med 2008; 12: 2189–2204. 8. Aravalli RN, Cressman EN, Steer CJ. Cellular and molecular mechanisms of
hepatocellular carcinoma: an update. Arch Toxicol 2013; 87: 227–247.
9. Guichard C, Amaddeo G, Imbeaud S, Ladeiro Y, Pelletier L, Maad IB et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet 2012; 44: 694–698.
10. Cleary SP, Jeck WR, Zhao X, Chen K, Selitsky SR, Savich GL et al. Identification of driver genes in hepatocellular carcinoma by exome sequencing. Hepatology 2013; 58: 1693–1702.
Table 2 miRNA-based therapies in HCC preclinical models
miRNAs Mouse model Method Delivery system
miR-221 Orthotopic model MiRNA inhibition Cholesterol-conjugated anti-miR-221 miR-221 TG-221 MiRNA inhibition Anti-miRNA oligonucleotide miR-494 tet-o-MYC;LAP-tTA MiRNA inhibition Anti-miRNA oligonucleotide miR-101 Xenograft MiRNA replacement RNA-duplex
miR-29b Xenograft MiRNA replacement 20-O-methyl-modified oligoribonucleotides
miR-199a/b-3p Xenograft MiRNA replacement Adeno-associated virus miR-199a/b-3p Xenograft MiRNA replacement Lentivirus
miR-375 Xenograft MiRNA replacement Cholesterol-conjugated 20-O-methyl-modified miRNA
miR-26a tet-o-MYC;LAP-tTA MiRNA replacement Adeno-associated virus miR-122 tet-o-MYC;LAP-tTAT; MiRNA replacement Adeno-associated virus miR-122 Xenograft MiRNA replacement Cationic lipid nanoparticles miR-124 DEN-induced HCC mouse model MiRNA replacement Liposomes
Let-7 Xenograft Oncolytic virus Adenovirus miR-122 Xenograft Oncolytic virus Adenovirus miR-122;
miR-124; let-7
Xenograft Oncolytic virus Herpes simplex virus miR-199 Xenograft; TG-221 Oncolytic virus Adenovirus
11. Kan Z, Zheng H, Liu X, Li S, Barber TD, Gong Z et al. Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma. Genome Res 2013; 23: 1422–1433.
12. Villanueva A, Llovet JM. Targeted therapies for hepatocellular carcinoma. Gastroenterology 2011; 140: 1410–1426.
13. Faivre S, Bouattour M, Raymond E. Novel molecular therapies in hepatocellular carcinoma. Liver Int 2011; 31(Suppl 1): 151–160.
14. Negrini M, Gramantieri L, Sabbioni S, Croce CM. microRNA involvement in hepatocellular carcinoma. Anticancer Agents Med Chem 2011; 11: 500–521.
15. Scarola M, Schoeftner S, Schneider C, Benetti R. miR-335 directly targets Rb1 (pRb/p105) in a proximal connection to p53-dependent stress response. Cancer Res 2010; 70: 6925–6933.
16. Tome M, Lopez-Romero P, Albo C, Sepulveda JC, Fernandez-Gutierrez B, Dopazo A et al. miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells. Cell Death Differ 2010; 18: 985–995.
17. Lupini L, Bassi C, Ferracin M, Bartonicek N, D’Abundo L, Zagatti B et al. miR-221 affects multiple cancer pathways by modulating the level of hundreds messenger RNAs. Front Genet 2013; 4: 64.
18. Fornari F, Gramantieri L, Ferracin M, Veronese A, Sabbioni S, Calin GA et al. MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma. Oncogene 2008; 27: 5651–5661.
19. Xu XR, Huang J, Xu ZG, Qian BZ, Zhu ZD, Yan Q et al. Insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver. Proc Natl Acad Sci USA 2001; 98: 15089–15094.
20. Joo M, Kang YK, Kim MR, Lee HK, Jang JJ. Cyclin D1 overexpression in hepatocellular carcinoma. Liver 2001; 21: 89–95.
21. Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F et al. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 2001; 120: 1763–1773.
22. Matsuda Y, Ichida T, Matsuzawa J, Sugimura K, Asakura H. p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology 1999; 116: 394–400.
23. Furuta M, Kozaki KI, Tanaka S, Arii S, Imoto I, Inazawa J. miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma. Carcinogenesis 2010; 31: 766–776.
24. Xu T, Zhu Y, Xiong Y, Ge YY, Yun JP, Zhuang SM. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 2009; 50: 113–121.
25. Ji J, Shi J, Budhu A, Yu Z, Forgues M, Roessler S et al. MicroRNA expression, survival, and response to interferon in liver cancer. N Engl J Med 2009; 361: 1437–1447.
26. Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009; 137: 1005–1017.
27. Ito Y, Takeda T, Sakon M, Tsujimoto M, Monden M, Matsuura N. Expression of p57/Kip2 protein in hepatocellular carcinoma. Oncology 2001; 61: 221–225.
28. Schwienbacher C, Gramantieri L, Scelfo R, Veronese A, Calin GA, Bolondi L et al. Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas. Proc Natl Acad Sci USA 2000; 97: 5445–5449.
29. Li Y, Tan W, Neo TW, Aung MO, Wasser S, Lim SG et al. Role of the miR-106b-25 microRNA cluster in hepatocellular carcinoma. Cancer Sci 2009; 100: 1234–1242.
30. Petrocca F, Visone R, Onelli MR, Shah MH, Nicoloso MS, de Martino I et al. E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 2008; 13: 272–286.
31. Carraro G, El-Hashash A, Guidolin D, Tiozzo C, Turcatel G, Young BM et al. miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution. Dev Biol 2009; 333: 238–250.
32. O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005; 435: 839–843.
33. Taguchi A, Yanagisawa K, Tanaka M, Cao K, Matsuyama Y, Goto H et al. Identification of hypoxia-inducible factor-1 alpha as a novel target for miR-17-92 microRNA cluster. Cancer Res 2008; 68: 5540–5545.
34. Connolly E, Melegari M, Landgraf P, Tchaikovskaya T, Tennant BC, Slagle BL et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype. Am J Pathol 2008; 173: 856–864.
35. Fabregat I, Roncero C, Fernandez M. Survival and apoptosis: a dysregulated balance in liver cancer. Liver Int 2007; 27: 155–162.
36. Guo XZ, Shao XD, Liu MP, Xu JH, Ren LN, Zhao JJ et al. Effect of bax, bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma. World J Gastroenterol 2002; 8: 1059–1062.
37. Lin CJ, Gong HY, Tseng HC, Wang WL, Wu JL. miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines. Biochem Biophys Res Commun 2008; 375: 315–320.
38. Shimizu S, Takehara T, Hikita H, Kodama T, Miyagi T, Hosui A et al. The let-7 family of microRNAs inhibits Bcl-xL expression and potentiates sorafenib-induced apoptosis in human hepatocellular carcinoma. J Hepatol 2010; 52: 698–704.
39. Su H, Yang JR, Xu T, Huang J, Xu L, Yuan Y et al. MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity. Cancer Res 2009; 69: 1135–1142.
40. Braconi C, Huang N, Patel T. MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes. Hepatology 2010; 51: 881–890.
41. Mott JL, Kobayashi S, Bronk SF, Gores GJ. mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene 2007; 26: 6133–6140.
42. Xiong Y, Fang JH, Yun JP, Yang J, Zhang Y, Jia WH et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 2010; 51: 836–845.
43. Gramantieri L, Fornari F, Ferracin M, Veronese A, Sabbioni S, Calin GA et al. MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality. Clin Cancer Res 2009; 15: 5073–5081.
44. Schmelzle T, Mailleux AA, Overholtzer M, Carroll JS, Solimini NL, Lightcap ES et al. Functional role and oncogene-regulated expression of the BH3-only factor Bmf in mammary epithelial anoikis and morphogenesis. Proc Natl Acad Sci USA 2007; 104: 3787–3792.
45. Padua D, Massague J. Roles of TGFbeta in metastasis. Cell Res 2009; 19: 89–102. 46. Fu H, Tie Y, Xu C, Zhang Z, Zhu J, Shi Y et al. Identification of human fetal liver miRNAs
by a novel method. FEBS Lett 2005; 579: 3849–3854.
47. Veronese A, Lupini L, Consiglio J, Visone R, Ferracin M, Fornari F et al. Oncogenic role of miR-483-3p at the IGF2/483 locus. Cancer Res 2010; 70: 3140–3149.
48. Saini S, Majid S, Yamamura S, Tabatabai ZL, Suh SO, Shahryari V et al. Regulatory role of miR-203 in prostate cancer progression and metastasis. Clin Cancer Res 2010. 49. Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N et al. MicroRNA
expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 2008; 299: 425–436.
50. Li R, You N, Wang X, Dou K. MicroRNAs involved in neoplastic transformation of liver cancer stem cells. J Exp Clin Cancer Res 2010; 29: 169.
51. Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT et al. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarci-noma cell lines. Gastroenterology 2006; 130: 2113–2129.
52. Blower PE, Chung JH, Verducci JS, Lin S, Park JK, Dai Z et al. MicroRNAs modulate the chemosensitivity of tumor cells. Mol Cancer Ther 2008; 7: 1–9.
53. Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S et al. MicroRNA-21 induces resistance to the anti-tumour effect of interferon-alpha/ 5-fluorouracil in hepatocellular carcinoma cells. Br J Cancer 2010; 103: 1617–1626.
54. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007; 133: 647–658.
55. Sayed D, He M, Hong C, Gao S, Rane S, Yang Z et al. MicroRNA-21 is a downstream effector of AKT that mediates its antiapoptotic effects via suppression of Fas ligand. J Biol Chem 2010; 285: 20281–20290.
56. Zhang JG, Wang JJ, Zhao F, Liu Q, Jiang K, Yang GH. MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in non-small cell lung cancer (NSCLC). Clin Chim Acta 2010; 411: 846–852.
57. Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, Post S et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 2008; 27: 2128–2136.
58. Chen Y, Liu W, Chao T, Zhang Y, Yan X, Gong Y et al. MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G. Cancer Lett 2008; 272: 197–205.
59. Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem 2008; 283: 1026–1033.
60. Lu Z, Liu M, Stribinskis V, Klinge CM, Ramos KS, Colburn NH et al. MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene. Oncogene 2008; 27: 4373–4379.
61. Motoyama K, Inoue H, Mimori K, Tanaka F, Kojima K, Uetake H et al. Clinicopathological and prognostic significance of PDCD4 and microRNA-21 in human gastric cancer. Int J Oncol 2010; 36: 1089–1095.
62. Selaru FM, Olaru AV, Kan T, David S, Cheng Y, Mori Y et al. MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3. Hepatology 2009; 49: 1595–1601. 63. Bressac B, Kew M, Wands J, Ozturk M. Selective G to T mutations of p53 gene in
hepatocellular carcinoma from southern Africa. Nature 1991; 350: 429–431. 64. Lu W, Lo SY, Chen M, Wu K, Fung YK, Ou JH. Activation of p53 tumor suppressor by
hepatitis C virus core protein. Virology 1999; 264: 134–141.
65. Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67: 6092–6099.
66. Fornari F, Gramantieri L, Giovannini C, Veronese A, Ferracin M, Sabbioni S et al. MiR-122/cyclin G1 interaction modulates p53 activity and affects doxorubicin sensitivity of human hepatocarcinoma cells. Cancer Res 2009; 69: 5761–5767.
67. Spizzo R, Nicoloso MS, Lupini L, Lu Y, Fogarty J, Rossi S et al. miR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor-alpha in human breast cancer cells. Cell Death Differ 2010; 17: 246–254.
68. Pineau P, Volinia S, McJunkin K, Marchio A, Battiston C, Terris B et al. miR-221 overexpression contributes to liver tumorigenesis. Proc Natl Acad Sci USA 2010; 107: 264–269.
69. Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 2007; 26: 745–752.
70. He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y et al. A microRNA component of the p53 tumour suppressor network. Nature 2007; 447: 1130–1134.
71. He X, He L, Hannon GJ. The guardian’s little helper: microRNAs in the p53 tumor suppressor network. Cancer Res 2007; 67: 11099–11101.
72. Hermeking H. p53 enters the microRNA world. Cancer Cell 2007; 12: 414–418. 73. Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N et al.
Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 2007; 26: 731–743.
74. Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A et al. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 2007; 6: 1586–1593.
75. Sachdeva M, Zhu S, Wu F, Wu H, Walia V, Kumar S et al. p53 represses c-Myc through induction of the tumor suppressor miR-145. Proc Natl Acad Sci USA 2009; 106: 3207–3212.
76. Georges SA, Biery MC, Kim SY, Schelter JM, Guo J, Chang AN et al. Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215. Cancer Res 2008; 68: 10105–10112.
77. Braun CJ, Zhang X, Savelyeva I, Wolff S, Moll UM, Schepeler T et al. p53-Responsive micrornas 192 and 215 are capable of inducing cell cycle arrest. Cancer Res 2008; 68: 10094–10104.
78. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003; 4: 915–925.
79. Benvenuti S, Comoglio PM. The MET receptor tyrosine kinase in invasion and metastasis. J Cell Physiol 2007; 213: 316–325.
80. Boix L, Rosa JL, Ventura F, Castells A, Bruix J, Rodes J et al. c-met mRNA overexpression in human hepatocellular carcinoma. Hepatology 1994; 19: 88–91. 81. Suzuki K, Hayashi N, Yamada Y, Yoshihara H, Miyamoto Y, Ito Y et al. Expression
of the c-met protooncogene in human hepatocellular carcinoma. Hepatology 1994; 20: 1231–1236.
82. Grigioni WF, Fiorentino M, D’Errico A, Ponzetto A, Crepaldi T, Prat M et al. Overexpression of c-met protooncogene product and raised Ki67 index in hepatocellular carcinomas with respect to benign liver conditions. Hepatology 1995; 21: 1543–1546.
83. Ueki T, Fujimoto J, Suzuki T, Yamamoto H, Okamoto E. Expression of hepatocyte growth factor and its receptor, the c-met proto-oncogene, in hepatocellular carcinoma. Hepatology 1997; 25: 619–623.
84. Fornari F, Milazzo M, Chieco P, Negrini M, Calin GA, Grazi GL et al. MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells. Cancer Res 2010; 70: 5184–5193.
85. Kim S, Lee UJ, Kim MN, Lee EJ, Kim JY, Lee MY et al. MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2). J Biol Chem 2008; 283: 18158–18166.
86. Li N, Fu H, Tie Y, Hu Z, Kong W, Wu Y et al. miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells. Cancer Lett 2009; 275: 44–53.
87. Salvi A, Sabelli C, Moncini S, Venturin M, Arici B, Riva P et al. MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells. FEBS J 2009; 276: 2966–2982.
88. Datta J, Kutay H, Nasser MW, Nuovo GJ, Wang B, Majumder S et al. Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res 2008; 68: 5049–5058.
89. Jagirdar J, Nonomura A, Patil J, Thor A, Paronetto F. ras oncogene p21 expression in hepatocellular carcinoma. J Exp Pathol 1989; 4: 37–46.
90. Challen C, Guo K, Collier JD, Cavanagh D, Bassendine MF. Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas. J Hepatol 1992; 14: 342–346.
91. Akao Y, Nakagawa Y, Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cells. Biol Pharm Bull 2006; 29: 903–906.
92. Bussing I, Slack FJ, Grosshans H. let-7 microRNAs in development, stem cells and cancer. Trends Mol Med 2008; 14: 400–409.
93. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A et al. RAS is regulated by the let-7 microRNA family. Cell 2005; 120: 635–647.
94. Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes Dev 2007; 21: 1025–1030.
95. Mayr C, Hemann MT, Bartel DP. Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 2007; 315: 1576–1579.
96. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004; 64: 3753–3756.
97. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007; 131: 1109–1123.
98. Liu C, Yu J, Yu S, Lavker RM, Cai L, Liu W et al. MicroRNA-21 acts as an oncomir through multiple targets in human hepatocellular carcinoma. J Hepatol 2010; 53: 98–107. 99. Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A et al. miR-221&222
regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell 2009; 16: 498–509.
100. Connolly EC, Van Doorslaer K, Rogler LE, Rogler CE. Overexpression of miR-21 promotes an in vitro metastatic phenotype by targeting the tumor suppressor RHOB. Mol Cancer Res 2010; 8: 691–700.
101. Gabriely G, Wurdinger T, Kesari S, Esau CC, Burchard J, Linsley PS et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol 2008; 28: 5369–5380.
102. Song G, Sharma AD, Roll GR, Ng R, Lee AY, Blelloch RH et al. MicroRNAs control hepatocyte proliferation during liver regeneration. Hepatology 2010; 51: 1735–1743. 103. Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY. MicroRNA-21 targets tumor suppressor
genes in invasion and metastasis. Cell Res 2008; 18: 350–359.
104. Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology 2008; 135: 1972–1983; 1983 e1-11.
105. Zhou L, Huang Y, Li J, Wang Z. The mTOR pathway is associated with the poor prognosis of human hepatocellular carcinoma. Med Oncol 2010; 27: 255–261.
106. Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ et al. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res 2008; 14: 419–427.
107. Shen Q, Cicinnati VR, Zhang X, Iacob S, Weber F, Sotiropoulos GC et al. Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion. Mol Cancer 2010; 9: 227.
108. Ichimi T, Enokida H, Okuno Y, Kunimoto R, Chiyomaru T, Kawamoto K et al. Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int J Cancer 2009; 125: 345–352.
109. Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P et al. MicroRNA signatures in human ovarian cancer. Cancer Res 2007; 67: 8699–8707.
110. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007; 129: 1401–1414.
111. Dolganiuc A, Petrasek J, Kodys K, Catalano D, Mandrekar P, Velayudham A et al. MicroRNA expression profile in Lieber-DeCarli diet-induced alcoholic and methionine choline deficient diet-induced nonalcoholic steatohepatitis models in mice. Alcohol Clin Exp Res 2009; 33: 1704–1710.
112. Chao A, Tsai CL, Wei PC, Hsueh S, Chao AS, Wang CJ et al. Decreased expression of microRNA-199b increases protein levels of SET (protein phosphatase 2A inhibitor) in human choriocarcinoma. Cancer Lett 2010; 291: 99–107.
113. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 2009; 28: 3526–3536.
114. Tsai WC, Hsu PW, Lai TC, Chau GY, Lin CW, Chen CM et al. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 2009; 49: 1571–1582.
115. Wong CC, Wong CM, Tung EK, Au SL, Lee JM, Poon RT et al. The MicroRNA miR-139 Suppresses Metastasis and Progression of Hepatocellular Carcinoma by Down-regulating Rho-Kinase 2. Gastroenterology 2011; 140: 322–331.
116. Ding J, Huang S, Wu S, Zhao Y, Liang L, Yan M et al. Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA. Nat Cell Biol 2010; 12: 390–399.
117. Polakis P. Wnt signaling and cancer. Genes Dev 2000; 14: 1837–1851.
118. Fodde R, Brabletz T. Wnt/beta-catenin signaling in cancer stemness and malignant behavior. Curr Opin Cell Biol 2007; 19: 150–158.
119. Vermeulen L, De Sousa EMF, van der Heijden M, Cameron K, de Jong JH, Borovski T et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol 2010; 12: 468–476.
120. Wend P, Holland JD, Ziebold U, Birchmeier W. Wnt signaling in stem and cancer stem cells. Semin Cell Dev Biol 2010; 21: 855–863.
121. Thompson MD, Monga SP. WNT/beta-catenin signaling in liver health and disease. Hepatology 2007; 45: 1298–1305.
122. Satoh S, Daigo Y, Furukawa Y, Kato T, Miwa N, Nishiwaki T et al. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet 2000; 24: 245–250.
123. Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A et al. Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin. Cells Tissues Organs 2005; 179: 56–65.
